In 2001, academic research labs may have been the first to introduce QD optical coding technology for contemplated uses in biological applications [see, for example, M. Han, X. Gao, J. Z. Su, S. Nie, Nat. Biotech., 2001, 19, 631]. Thus far, however, commercial applications based on the previous work of such academic research labs may have been somewhat slow to develop.
Earlier excitement over the potential biological applications for such QD optical coding (or QD “barcoding”) technology may have stemmed, to some degree, from the prospect of making highly sensitive measurements of multiple protein and/or nucleic acid targets on a substantially simultaneous basis. [See, for example: H. Xu, M. Sha, E. Y. Wong, J. Uphoff, Y. Xu, J. A. Treadway, A. Truong, E. O'Brien, S. Asquith, M. Stubbins, N. K. Spurr, E. H. Lai, W. Mahoney, Nucleic Acids Research, 2003, 31, e43; and Y. Ho, M. C. Kung, S. Yang, T. Wang, Nano Lett., 2005, 5, 1693.] It may also have been believed that, in comparison to optical signals emitted by organic fluorophores, the use of QDs might enable the generation and use of a greater number of optical codes due to their substantially tunable emission and/or relatively narrow spectral linewidth. [See, for example: M. Bruchez, M. Moronne, P. Gin, S. Weiss, A_P. Alivisatos, Science, 1998, 281, 2013; W. C. W. Chan, S. Nie, Science, 1998, 281, 2016; A. P. Alivisatos, J. Phys. Chem., 1996, 100, 13226; I. L. Medintz, H. T. Uyeda, E. R. Goldman, H. Mattoussi, Nat. Mater., 2005, 4, 435; and J. M. Klostranec, W. C. W. Chan, Adv. Mat., 2006, 18, 1953.] QD optical codes may additionally have been thought to require only a low-power single excitation source, due to their large absorption cross-section and continuous absorption profile—a factor which might have led to significant reductions in the complexity and cost of instrumentation. Indeed, it may have been suggested that such a system could rival microarray technologies, which are commonly used for measuring large numbers of biological molecules in a short period of time. [See, for example: R. F. Service, Science, 1998, 282, 396; D. Gershon, Nature, 2005, 437, 1195; and M. Eisenstein, Nature, 2006, 444, 959.]
Much effort in recent years may have been focused on the reproducible synthesis of QD optical codes. [See, for example: X. Gao, S. Nie, Anal. Chem., 2004, 76, 2406; T. R. Sathe, A. Agrawal, S. Nie, Anal. Chem., 2006, 78, 5627; and N. Gaponik, I. L. Radtchenko, G. B. Sukhorukov, H. Weller, A. L. Rogach, Adv. Mat., 2002, 14, 879.]
Previously, however, the method of reading QD optical codes and the potential impact of environmental conditions on fluorescence stability of the optical codes may have been largely ignored. Though not essential to the working of the present invention, it may now be believed that, without considering these two constraints, the accurate identification of QD optical codes might be compromised, potentially leading to false assay outcomes. There may, therefore, be a need for a rapid, reliable and accurate method of deconvolving fluorescence spectra generated by QD optical coding technology, in which there is often a large degree of spectral overlap. In addition to signal deconvolution, there is also a need for a method of identifying the QD optical codes that takes into account the impact of environmental factors on fluorescence stability.
It is an object of one preferred embodiment according to the invention to provide a system and/or method for creating, and/or selecting a set of, optical codes (preferably, but not necessarily, QD optical codes) with unique spectral signatures.
It is an object of one preferred embodiment according to the invention to provide a system and/or method for Gaussian curve modeling of single-color fluorophores (preferably, but not necessarily, QDs), preferably but not necessarily for reference use in deconvolving one or more QD optical codes.
It is an object of one preferred embodiment according to the invention to provide for the relatively rapid and/or substantially accurate read-out of QD optical codes.
It is an object of one preferred embodiment according to the invention to provide a system and/or method for making substantially accurate estimates of local peak information for QD optical codes.
It is an object of one preferred embodiment according to the invention to minimize or reduce the chances of falsely identifying QD optical codes.
It is an object of one preferred embodiment according to the invention to provide a system and/or method adapted to deconvolute mixed fluorescence spectral signals in optical codes and/or in mixtures of fluorophores (preferably, but not necessarily, mixtures of QDs).
It is an object of one preferred embodiment according to the invention to provide a system and/or method adapted to deconvolute mixed fluorescence spectral signals composed of one or more, and preferably at least two, unique QD emissions in solution.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that, after deconvolution of a mixed fluorescence spectral signal, generates deconvolved spectra with similar appearance and/or emission wavelengths to single-color QD spectra.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that accurately deconvolves mixed fluorescence spectral signals that possess a large degree of overlap in their component single-color fluorescence spectra.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that may be generalized for use with QD optical codes composed of more than two colors.
It is an object of one preferred embodiment according to the invention to provide a system and/or method for signal deconvolution that accounts, to some degree, for instrumental measurement errors and/or for a specific decoding scheme utilized.
It is an object of one preferred embodiment according to the invention to provide a system and/or method for screening out ambiguous QD combinations from a set of selected QD optical codes.
It is an object of one preferred embodiment according to the invention to provide a set of QD optical codes that is advantageously selected to balance accurate read-out with a high number of usable barcodes.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that minimizes or reduces risks associated with measurement error.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that is adapted for use in situations where microbead size and/or doping yields cannot be precisely controlled.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that enables selection of a relatively reliable detection scheme based on known sources and/or patterns of signal variation amongst QD optical codes.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that is adaptable for use in different environmental conditions, such as, for example, in varying storage buffer types and pHs.
It is an object of one preferred embodiment according to the invention to provide a system and/or method that is adaptable to be used with QDs of various sizes.
It is an object of one preferred embodiment according to the invention to provide a system and/or method for use in biological and/or medical applications.
It is an object of the present invention to obviate or mitigate one or more of the aforementioned mentioned disadvantages associated with the prior art, and/or to achieve one or more of the aforementioned objects of the invention.